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At least 1,027 records · Page 57Linked to original sources
PubMed2003

Analysis of transcriptional regulatory sequences in the human endogenous retrovirus W long terminal repeat.

The U3 region of the human endogenous retrovirus W long terminal repeat (HERV-W LTR) contains several putative regulatory sequences that might not only regulate transcription of viral genes but also influence the expression of neighbouring cellular genes. In this study, we analysed the U3 region in detail in order to understand the transcriptional regulatory mechanism of HERV-W. Two transcription factor (TF) binding sites for Oct-1 and C/EBP were important as a silencer and an enhancer, respectively, for transcriptional regulation. Furthermore, it was possible to divide the HERV-W LTR isolates into two groups depending on their promoter strength, which might be determined by the integrity of the two TF binding sites. However, neither the Oct-1 binding site nor the CAAT-box was required for the cell type-specific activity of the HERV-W LTR. Instead, the 3' terminus of U3 from 191 to 260, which includes a TATA box, was sufficient for specificity, suggesting that the efficiency of assembly of basic transcription machinery at the TATA box of HERV-W LTR might determine the cell type specificity.

Binding Sites↗
PubMed1999

A human TATA binding protein-related protein with altered DNA binding specificity inhibits transcription from multiple promoters and activators.

The TATA binding protein (TBP) plays a central role in eukaryotic and archael transcription initiation. We describe the isolation of a novel 23-kDa human protein that displays 41% identity to TBP and is expressed in most human tissue. Recombinant TBP-related protein (TRP) displayed barely detectable binding to consensus TATA box sequences but bound with slightly higher affinities to nonconsensus TATA sequences. TRP did not substitute for TBP in transcription reactions in vitro. However, addition of TRP potently inhibited basal and activated transcription from multiple promoters in vitro and in vivo. General transcription factors TFIIA and TFIIB bound glutathione S-transferase-TRP in solution but failed to stimulate TRP binding to DNA. Preincubation of TRP with TFIIA inhibited TBP-TFIIA-DNA complex formation and addition of TFIIA overcame TRP-mediated transcription repression. TRP transcriptional repression activity was specifically reduced by mutations in TRP that disrupt the TFIIA binding surface but not by mutations that disrupt the TFIIB or DNA binding surface of TRP. These results suggest that TFIIA is a primary target of TRP transcription inhibition and that TRP may modulate transcription by a novel mechanism involving the partial mimicry of TBP functions.

Amino Acid Sequence↗
PubMedDate not supplied

Light regulation of plant gene expression by an upstream enhancer-like element.

Light regulates many varied physiological and developmental phenomena during plant growth and differentiation, including the formation of a photosynthetically competent chloroplast from a proplastid. The expression of ribulose 1,5-bisphosphate carboxylase small subunit (rbcS) genes is regulated by light in a development- and tissue-specific manner2,3. In some plant species, phytochrome has been demonstrated to mediate this response, and photoregulation of rbcS expression occurs at least in part at the level of transcription. We have shown previously that a 5'-noncoding fragment (4-973 base pairs (bp) upstream of the messenger RNA cap site) of the pea rbcS ss3.6 gene contains all of the nucleotide sequence information necessary to direct the photoregulated expression of a bacterial chloramphenicol acetyltransferase (cat) gene in tobacco. Consistent with these findings, Morelli et al.11 have shown by deletion analysis of a second rbcS gene promoter, that the sequences required for photoregulated expression of rbcS E9 reside within the 5'-noncoding region. They identified an upstream region of approximately 700 bp needed for maximum transcription but not light-dark regulation, and a region from -35 to -2 bp which included the TATA box and contained the necessary information for light responsiveness. We now demonstrate that regulatory sequences 5' distal to the rbcS ss3.6 TATA box and transcriptional start site not only contain the information necessary for maximum expression, but also confer photoregulation. These upstream regulatory sequences function independently of orientation when fused to their homologous promoter or a heterologous promoter.

Acetyltransferases↗
PubMed1989

Novel regulation of transcription initiation of the peptide IX gene of adenovirus 2.

cis-Acting elements involved in transcription of the peptide IX (pIX) gene of adenovirus 2 were identified by using in vivo transient expression assays and two in vitro transcription systems. Deletion of either the sequence between positions -45 and -70 or the TATA box abolished the initiation of pIX gene transcription in vivo and transcription with HeLa cell nuclear extracts in vitro. These results initially suggested the presence of a positive factor acting on the upstream element. However, when proteins in the nuclear extract were fractionated by column chromatography and analyzed by reconstitution of transcription in vitro, it was found that a certain fraction could direct TATA box-dependent transcription initiation even in the absence of the upstream element. Furthermore, activity inhibiting TATA box-dependent transcription was found in the nuclear extract. In contrast, inhibition of TATA box-dependent transcription was suppressed by deletion of a downstream sequence between positions +33 and +122. These results indicate that the TATA box of the pIX gene by itself has the ability to direct initiation of constitutive transcription but that the function of this element is under negative control by a repressor acting on a downstream sequence. Thus, the upstream element of the pIX gene appears to have a novel function: suppression of the transcriptional repression exerted by a downstream sequence, leading to a net transcription activation. Possible mechanisms for transcription initiation of pIX DNA are discussed.

Adenoviruses, Human↗
PubMed2002

A TATA binding protein mutant with increased affinity for DNA directs transcription from a reversed TATA sequence in vivo.

The TATA-binding protein (TBP) nucleates the assembly and determines the position of the preinitiation complex at RNA polymerase II-transcribed genes. We investigated the importance of two conserved residues on the DNA binding surface of Saccharomyces cerevisiae TBP to DNA binding and sequence discrimination. Because they define a significant break in the twofold symmetry of the TBP-TATA interface, Ala100 and Pro191 have been proposed to be key determinants of TBP binding orientation and transcription directionality. In contrast to previous predictions, we found that substitution of an alanine for Pro191 did not allow recognition of a reversed TATA box in vivo; however, the reciprocal change, Ala100 to proline, resulted in efficient utilization of this and other variant TATA sequences. In vitro assays demonstrated that TBP mutants with the A100P and P191A substitutions have increased and decreased affinity for DNA, respectively. The TATA binding defect of TBP with the P191A mutation could be intragenically suppressed by the A100P substitution. Our results suggest that Ala100 and Pro191 are important for DNA binding and sequence recognition by TBP, that the naturally occurring asymmetry of Ala100 and Pro191 is not essential for function, and that a single amino acid change in TBP can lead to elevated DNA binding affinity and recognition of a reversed TATA sequence.

Adenosine Triphosphatases↗
PubMed1986

Activation of the Drosophila hsp27 promoter by heat shock and by ecdysone involves independent and remote regulatory sequences.

Transcription of the Drosophila hsp27 gene is induced both by heat shock and by the steroid hormone ecdysone. Deletion analysis of the promoter using a transient expression assay in Drosophila tissue culture cells reveals two separate and functionally independent regulatory regions, whose positions coincide with previously reported DNase I-hypersensitive sites in embryonic chromatin. Ecdysone induction is mediated by multiple elements between -579 and -455. Several matches to the consensus heat shock regulatory element (HSE) lie between -370 and -270, and deletion of these abolishes the response to heat shock. Heat inducibility can be restored by the addition of HSE sequences from the hsp70 promoter, even when these are placed at the 3' end of the gene. Furthermore, HSEs seperated by >2 kb can interact in a cooperative way to promote transcription. Deletion of sequences adjacent to the TATA box (-40 to -154) has little effect on induction by either heat shock or ecdysone. In this respect, activation of the hsp27 TATA box in Drosophila cells differs from the long-range activation of promoters by HSEs or viral enhancers in HeLa cells.

Journal Article↗
PubMed1996

The BARE-1 retrotransposon is transcribed in barley from an LTR promoter active in transient assays.

The BARE-1 retrotransposon occurs in more than 10(4) copies in the barley genome. The element is bounded by long terminal repeats (LTRs, 1829 bp) containing motifs typical of retrotransposon promoters. These, the presence of predicted priming sites for reverse transcription, and the high conservation for all key functional domains of the coding region suggest that copies within the genome could be active retrotransposons. In view of this, we looked for transcription of BARE-1 within barley tissues and examined the promoter function of the BARE-1 LTR. We demonstrate here that BARE-1-like elements are transcribed in barley tissues, and that the transcripts begin within the BARE-1 LTR downstream of TATA boxes. The LTR can drive expression of reporter genes in transiently transformed barley protoplasts. This is dependent on the presence of a TATA box functional in planta as well. Furthermore, we identify regions within the LTR responsible for expression within protoplasts by deletion analyses of LTR-luc constructs. Similarities between promoter regulatory motifs and regions of the LTR were identified by comparisons to sequence libraries. The activity of the LTR as a promoter, combined with the abundance of BARE-1 in the genome, suggests that BARE-1 may retain the potential for propagation in the barley genome.

Base Sequence↗
PubMed1995

cis-acting elements and transcription factors involved in the promoter activity of the human factor VIII gene.

Factor VIII is a glycoprotein that is essential for blood coagulation. Although factor VIII mRNA has been detected in a variety of human tissues, hepatocytes are considered to be the major source of plasma factor VIII. In this report we demonstrate that the 5'-flanking region of the factor VIII gene is able to transcribe a luciferase reporter gene in three human liver-derived cell lines: PLC/PRF/5, Chang, and HepG2. DNase I footprinting showed the presence of 19 protein binding sites (labeled A to S, proximal to distal) distributed along the region from nucleotide -1175 to -9 of the factor VIII promoter (+1 refers to the translation initiation codon, ATG). Functional analysis of 5' and 3' deletion mutants of the promoter region in PLC/PRF/5 cells revealed that the region from -279 to -64, including sites B to D, contains all the necessary elements for maximal promoter activity. By using electrophoretic mobility shift assays with nuclear extracts and purified transcription factors, and antibody supershift assays we were able to characterize four liver-enriched factors and one ubiquitous transcription factor interacting with the proximal promoter binding sites (sites A to E): hepatocyte nuclear factor (HNF) 1 (site A), NF kappa B (site B), C/EBP alpha and C/EBP beta (proximal and distal regions of site C, and site D), and HNF4 (site E). Additionally, mutation of the putative TATA box GATAAA (positions -201 to -196) to GACCGA resulted in less than 2-fold decrease in promoter activity, suggesting that the putative TATA box is not essential for factor VIII promoter activity. These results significantly contribute to the understanding of the control of the hepatic transcription of the factor VIII gene.

Animals↗
PubMed1997

Cloning of the functional promoter for human insulin-like growth factor binding protein-4 gene: endogenous regulation.

The majority of the colon cancers analyzed to-date express insulin-like growth factor binding protein (IGFBP)-4, and antisense inhibition of IGFBP-4 messenger RNA (mRNA) confers a growth advantage to the cells in response to endogenous and exogenous IGFs. We recently reported a significant up-regulation of IGFBP-4 expression in a human colon cancer cell line (CaCo2) on spontaneous differentiation of the cells in culture. This suggests that the expression of IGFBP-4 may be related to growth and differentiation of colon cancer cells. To study the endogenous factors involved in the transcriptional regulation of IGFBP-4, we have isolated and sequenced the human (h) IGFBP-4 promoter. The approximately 1.3 kilobase pair (kb) 5' flanking region of the IGFBP-4 gene is GC rich and possesses several potential regulatory elements. These elements include a typical TATA box with sequence TATAA, located -299 nt from the initiation ATG codon. The cap site is located 14 nt downstream of the TATA box as determined by primer extension analysis. A 1.4-kb DNA fragment including the 1.254 kb 5' flanking region of the hIGFBP-4 gene was subcloned into a luciferase reporter vector (pGL-2 basic) either in the sense (BP-4-S-pGL) (S) or antisense (BP-4-AS-pGL) (AS) (negative control) orientation, relative to the luciferase coding sequence in the vector. CaCo2 cells were transfected with either the S or the AS vectors on days 2-10 of culture; cotransfection with the SV40-beta-Galactidose (Gal) vector was used to correct for transfection efficiency. The ratio of luciferase/beta-Gal expression by CaCo2 cells transfected with the S vectors increased significantly from days 3 and 4 to days 5 and 6 of culture, followed by a sharp decline on days 7-9, resembling the pattern of endogenous expression of IGFBP-4 by the cells; the expression of luciferase by the AS vectors remained low and insignificant. These results thus suggest that the approximately 1.4 kb 5' flanking region of the IGFBP-4 gene contains the cis elements required for regulation of the IGFBP-4 gene. Cloning and sequencing of the functional hIGFBP-4 promoter will enable us, for the first time, to study the endogenous factors/mechanisms responsible for the growth/differentiation (cell density) associated regulation of IGFBP-4 expression in colonic epithelial cells.

Base Sequence↗
PubMed1997

Bovine insulin-like growth factor binding protein-3: organization of the chromosomal gene and functional analysis of its promoter.

Insulin-like growth factor binding protein-3 (IGFBP-3), the major IGFBP in the circulation, is synthesized by the vascular endothelium in vivo and has been shown to be an important modulator of the physiological effects of IGF. IGFBP-3 is regulated by a number of growth factors/cytokines to which the vascular endothelium is exposed, including IGF-I stimulation and TGF-beta1 inhibition of IGFBP-3 in cultured endothelial cells. To understand the mechanisms of transcriptional regulation of IGFBP-3, we have cloned the bovine IGFBP-3 gene and begun the functional analysis of its promoter. Southern analysis indicated a single copy gene. The gene spanned approximately 10 kb and was divided into five exons, the fifth containing the 3' untranslated region. The transcription start site was 137 bp upstream of the initiation codon and a TATA box was located 26 bp 5' to this CAP site. No CAAT box was present but a GC rich sequence element, containing two overlapping putative AP-2 binding elements, was located 5' to the TATA box. Transient transfection studies with a series of 5' truncated luciferase reporter constructs were conducted in primary cultures of bovine aorta endothelial cells. Results of the transfection studies indicated that 1) nearly 80% of the maximal basal promoter activity was retained within the first 130 bp of the 5' flanking sequence; 2) this region responded to IGF-I, despite lacking the TTF-1/TTF-2 (thyroid specific transcription factors) binding elements that are required for IGF-I stimulation of thyroglobulin synthesis. These binding elements have also been suggested to be involved in IGF-I regulation of IGFBP-3 transcription, thus, implying the existence of novel cis-acting elements that mediate the IGF-I stimulation of bovine endothelial cell IGFBP-3 mRNA synthesis; 3) deletion of the GC rich sequence element resulted in a 60% reduction in basal promoter activity as well as loss of the IGF-1 stimulatory effect; 4) the TGF-beta1 mediated inhibition of IGFBP-3 transcription required sequence element(s) beyond 1.5 kb of its promoter.

Animals↗
PubMed1988

Detection of specific beta-globin mutations in Kurdish Jews with beta-thalassemia.

Patients with beta-thalassemia, of Kurdish extraction, were screened for the presence of two mutations, in the TATA box and in codon 44, previously discovered in this ethnic isolate. Of the 56 chromosomes analyzed, 13 were found to carry the TATA box mutation and 17 the codon 44 mutation. The result of this work provides a basis for a more efficient prenatal diagnosis program for this community.

Adolescent↗
PubMed2005

Comparative genomics on mammalian Fgf3-Fgf4 locus.

The CCND1-ORAOV1-FGF19-FGF4-FGF3-TMEM16A-FADD-PPFIA1-CTTN (EMS1) locus at human chromosome 11q13.3 is amplified in head and neck tumors, esophageal cancer, Kaposi's sarcoma, bladder tumors, breast cancer, and liver cancer. Fgf4 mRNA is expressed in embryonic stem (ES) cells depending on Sox2 and Pou5f1 (Oct3/Oct4) transcription factors, and in myotomes and limb bud AER depending on MyoD (or Myf5) and GATA transcription factors. Here, rat Fgf3 and Fgf4 complete coding sequences were determined by using bioinformatics. Multiple errors, including one-base insertion and 22-base deletion, were identified within the coding region of rat Fgf4 RefSeq (NM_053809.1 or AB079673.1). Rat Fgf3 and Fgf4 genes, consisting of three exons, were clustered in tail-to-head manner with an interval of about 16 kb. CUTL1 (CCAAT-displacement protein, CDP) and NKX2-5 binding sites and TATA box within 5'-flanking promoter region were conserved among human, rat and mouse Fgf3 orthologs. MYOD and MYOG (Myogenin) binding sites and TATA box within 5'-flanking promoter region as well as GATA, MYOD, SOX2 and POU5F1 binding sites within exon 3 were conserved among mammalian Fgf4 orthologs. Human FGF3 and FGF4 genes were clustered in tail-to-head manner with an interval of about 35 kb. Major repetitive sequence (FGF34Rep1) and minor repetitive sequence (FGF34Rep2) were identified within human FGF3-FGF4 gene cluster. FGF34Rep1 were clustered within the FGF3-FGF4 locus as well as around the IL28RA locus (1p36.11) and the NFAM1 locus (22q13.2). FGF34Rep2 was characterized by the CCA(T/C) repeats. This is the first report on comparative genomics analyses on the Fgf3-Fgf4 locus within human, rat and mouse genomes.

Amino Acid Sequence↗
PubMed1993

Structure and promoter organization of the human monoamine oxidase A and B genes.

Monoamine oxidase (MAO) A and B play an important role in regulating levels of biogenic amines. MAO A and B cDNAs have been cloned and the deduced amino acids share 73% sequence identity. The genes for MAOA and B are comprised of 15 exons interspersed by 14 introns, span at least 60 kb and exhibit identical exon-intron organization. These findings suggest that the MAOA and MAOB genes are derived from the duplication of a common ancestral gene. The core promoter region of MAOA is comprised of two 90 bp repeats, each of which contains two Spl elements and lacks a TATA box. The MAOB core promoter region contains two sets of overlapping Spl sites which flank a CACCC element all upstream of a TATA box. The different organization of the MAOA and MAOB promoters may underlie their different cell and tissue specific expression.

Base Sequence↗
PubMed1984

Yeast RNA polymerase II initiates transcription in vitro at TATA sequences proximal to potential non-B forms of the DNA template.

Pure yeast RNA polymerase II selectively initiates an abortive in vitro transcript within a TATA box of the yeast iso-1 cytochrome c gene promoter. Using a series of promoter deletions we show that a DNA sequence located upstream of the TATA box is needed for an efficient in vitro transcription. Supercoiling of the DNA template is an absolute requirement for the specific in vitro transcription. Examination of the DNA structure near several in vitro initiation sites shows that the common features observed are the presence of a TATA sequence in which RNA synthesis is initiated, and which is proximal to a potential non-B form of the DNA (a B to Z transition or a cruciform structure).

Base Sequence↗
PubMed1994

Identification of multiple promoters within the N-ras proto-oncogene.

N-ras possesses a 'housekeeping' promoter, being G + C-rich and devoid of a TATA-box. Transcription initiates at a number of locations within this gene, a phenomena that is generally attributed to the absence of a TATA-box. In this report we investigate the possibility that multiple promoters, which could potentially contribute to the observed 5' end heterogeneity, exist within the murine N-ras gene. The 5' region of the gene was subdivided into several fragments, each corresponding to a region in which one or more transcription initiation site(s) had been mapped, and the ability of each fragment to express a reporter gene was assessed. Promoter activity was found associated with three independent, non-overlapping fragments, two of which were located entirely within transcribed regions of the gene. We found that these intragenic promoters were able to express the N-ras gene itself, as well as the reporter gene. In addition, we found that the activity of an intragenic promoter fragment was dependent upon the presence of regions encompassing initiation sites, and that a small fragment (approximately 40 bp) encompassing several initiation sites possessed promoter activity. These data support the existence of an 'initiator' element within the N-ras gene. Overall, our results demonstrate that multiple promoters reside within N-ras and suggest that they may play a role in generating the observed mRNA 5' end heterogeneity. The identification of multiple promoters within N-ras may have important implications regarding the regulation of expression of this gene in normal and malignant tissues. In addition, since a number of other genes with housekeeping promoters also initiate transcription at multiple locations, it is possible that the utilization of multiple promoters may represent a common feature of this class of genes.

Animals↗
PubMed1986

Characterization of cells transformed by the human polyomavirus JC virus.

One unique feature of the prototype JC virus (JCV) (Mad 1) genome is the occurrence of a second TATA sequence within the early promoter region. A naturally occurring oncogenic variant of JCV (Mad 4) lacks this second TATA box. Several cell lines transformed by Mad 1, Mad 4 and simian virus 40 were characterized, in part to investigate whether the second TATA sequence is functional. S1 nuclease mapping of early JCV gene transcription products revealed a major set of start sites common to both Mad 1 and Mad 4 mRNAs. In addition, a second set of early transcripts was found exclusively in Mad 1 transformants, presumably positioned by the second TATA box. The presence of these unique mRNAs in the Mad 1-transformed cells did not appear to have any bearing on the other parameters investigated, including size and quantity of early viral proteins, integration patterns of viral DNA and growth properties of the cells.

Antigens, Viral, Tumor↗
PubMed1992

Characterization of human cytomegalovirus UL84 early gene and identification of its putative protein product.

The DNA sequence and transcription pattern of human cytomegalovirus early gene UL84 were analyzed. This gene was mapped within a 2.6-kb PstI fragment located between 0.534 and 0.545 map unit of the large unique segment of the human cytomegalovirus genome, which is adjacent to the pp65 and pp71 genes. A 2.0-kb mRNA was transcribed from this region in the same leftward direction as the mRNAs of the pp65 and pp71 genes. The message was first detected at 2.5 h postinfection and reached a maximal level between 72 and 96 h postinfection. The nucleotide sequences of the 2.6-kb PstI genomic DNA fragment and the cDNA derived from this region were determined. The resulting data revealed a polyadenylation signal (AATAAA) located 14 nucleotides upstream from the poly(A) tail of the cDNA and a 1,761-bp open reading frame capable of encoding a 65-kDa polypeptide. A potential leucine zipper was found in the N-terminal half of the peptide molecule between amino acids 114 and 135. In addition, a different periodic leucine repeat with leucine at every eighth position was found between amino acids 325 and 373. The transcriptional initiation site of this early gene was determined by primer extension analysis. A putative TATA box (TATTTAA) located 24 bp upstream of the cap site and several inverted repeats were found in the region further upstream of the TATA box. To test whether the open reading frame of this cDNA encodes a virus-specific protein, the cDNA was overexpressed in Escherichia coli as a fusion protein used to generate antibodies in rabbits. A protein with a molecular size of 65 kDa was detected in the infected-cell extracts harvested at 6 to 72 h postinfection, but not in purified virions, using immunoblot analysis. Both nuclear and cytoplasmic fluorescences were found at late stages of virus infection. From the results obtained, we postulate that UL84 may be a stable, virus-specific, nonstructural protein capable of forming a homo- or heterodimeric molecule.

Amino Acid Sequence↗
PubMed1985

Structure of the rat alpha 1-acid glycoprotein gene.

The complete nucleotide sequence of the rat alpha 1-acid glycoprotein gene has been determined from an isolated lambda recombinant bacteriophage. Southern blot analysis and DNA sequencing indicate that there is only one gene per genome; it contains six exons and is located within a 3,200-base-pair fragment starting from a TATA box and extending to the polyadenylation signal AATAAA. Transcription starts 37 base pairs upstream from the beginning of the translation codon ATG. The TATA box (TATAAA) lies 26 base pairs upstream from this site. The gene contains several potential glucocorticoid receptor-binding sites, both inside and outside the structural gene.

Animals↗